Botanical Modulation of AhR-ER? by Crosstalk Inhibitors Promotes Estrogen (E2) Detoxification Estrogen receptor (ER) positive breast cancer poses significant health risks for postmenopausal women, and is in part mediated by the estrogen (E2) metabolite, estradiol-3,4-quinone, which causes depurinating adducts and leads to mutations. P450 1B1 is the primary enzyme for the conversion of estradiol to the 4-hydroxylated product, which can result in a genotoxic 4-quinone, while P450 1A1, which is normally epigenetically inhibited by E2-activated estrogen receptor alpha (ER?), converts estradiol to a nongenotoxic 2-hydroxylated product. Activated aryl hydrocarbon receptor (AhR) induces degradation of ER? as well as the transcription of both CYP1A1 and CYP1B1, which are translated into P450 1A1 and 1B1 respectively. Activators and ligands of AhR (Crosstalk Inhibitors) have been shown to preferentially upregulate CYP1A1 and ultimately the nongenotoxic 2-hydroxylated estradiol metabolite (estrogen detoxification pathway), through downregulation of the epigenetic inhibition of CYP1A1. Women have increasingly turned to botanical dietary supplements (BDS), instead of traditional hormone replacement therapy (HRT), since the release of the findings that estrogen + progesterone increases breast cancer risk. Interestingly, icaritin, a bioactive compound from Epimedium sp., a botanical used for women's health purposes, has been shown to activate AhR. The hypothesis of this proposal is that some women's health botanicals contain antiestrogenic, AhR activating compounds which degrade ER? (Aim 1) and reverse epigenetic CYP1A1 inhibition to preferentially activate the estrogen detoxification pathway in an in vitro cell culture model (Aim 2) and an in vivo ovariectomized rat model (Aim 3). Inhibition of estrogen-dependent alkaline phosphatase activity in Ishikawa cells, and a failure to inhibit a fluorescent estradiol-ER? complex by botanical compounds in a fluorescence polarization enzyme assay will provide antiestrogenic compounds which do not act through ER?. These compounds will be subjected to in- cell western of MCF-7 (ER+) cells against an ER? antibody to find compounds which degrade ER?. Additional XRE-luciferase activity in HepG2 cells will indicate AhR activating compounds, while a decrease in methylation through a ChIP assay of DNMT and XRE, and qRT-PCR of CYP1A1/1B1 will show that the reversal of estradiol mediated epigenetic CYP1A1 inhibition leads to upregulation of the estrogen detoxification pathway by Crosstalk Inhibitors in vitro. This premise will be taken to an ovariectomized rat model involving E2 and botanical compounds before sacrificing and analyzing uterine weight, performing LC-MS analysis of the E2 metabolites, and quantifying ER? expression levels using immunohistochemistry. This research may reveal mechanisms by which some bioactive women's health BDS compounds exhibit antiestrogenic outcomes, and the importance of the E2 detoxification pathway in promoting wellness in an in vivo postmenopausal model.